JP2000219637A - Erysipelothrix rhusiopathiae antigen composition and vaccine preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject vaccine composition that is useful as a vaccine for vaccinating animals, preferably mammalians or birds by using a fluid fraction originating from a specific culture mixture and stabilizers. SOLUTION: This vaccine composition comprises (A) a fluid fraction originating from the culture mixture of Erysipelothrix rhusiopathiae (swine erysipelas) and (B) a stabilizer. The component B is selected from metal hydroxides, metal phosphates, aluminum hydroxide gel, aluminum phosphate gel, calcium phosphate gel, zinc hydroxide/calcium hydroxide gel or alum. In the component B, the culture mixture is deactivated with formalin or β-propiolactone and the fraction is preferably concentrated in 3-30 times. For example, the aluminum hydroxide gel is added on the concentrate of the culture mixture so that the final concentration may reach about 10-40 vol.%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antigen composition and a vaccine preparation for preventing or suppressing Erysipelas infectious disease (Erysipelas) infection, and methods for preparing and using such an antigen composition and a vaccine preparation. About.

[0002]

BACKGROUND OF THE INVENTION Erysipelas are distributed worldwide and have become an economically significant problem throughout Europe, Asia, Australia and North and South America. 3 months to 3
Aged pigs are most susceptible to erysipelas. Affected pigs often have swollen and stiffened joints and do not gain weight efficiently. In addition, the carcasses are often removed or discarded by inspectors at processing plants.

About 10 years ago, from a completely sterilized culture, E. coli It has been shown that the practice of making Luciopathie vaccines is not necessary. Sterile filtrates also function to protect both pigs and mice against virulent loading. Subsequent published studies by Japanese and American scientists confirmed this finding, and Luciopathie contains all E. coli in the culture medium. It was shown to release antigen, a universal immunogen that immunizes pigs against Rusiopathiae strains (Sawada and Ta
kahashi, 1987, Am, J. Vet. Res. 48 : 239-242; Gros
chup et al., 1991, Epidemiol. Infect. 107: 637-649).
Groschup et al. Found that a 64-66 kDa protein in culture caused virulent E. coli in mice. It was shown to protect against the load of Luciopathie. Because such proteins have been shown to also protect pigs, USDA provides vaccine manufacturers with monoclonal antibodies (mAbs) to this protein for use in assays for this protein.

[0004] While vaccines that are effective in preventing erysipelas in pigs are highly desirable, none of the many traditional erysipelas vaccines provide acceptable protection against weaned pigs. The problem lies in the lack of sustained immunity. The pig industry needs a vaccine that, when fed at weaning, protects pigs against slaughter, ie, up to about six months of age against this deadly catastrophic disease. USDA has specified this requirement as a standard for licensing new vaccines.

[0005]

SUMMARY OF THE INVENTION The present invention relates to an E.C. The present invention relates to an antigen composition of Luciopathiae and a method for producing such an antigen composition. The invention further relates to E.C. The present invention relates to a vaccine preparation comprising an antigenic composition of Luciopathiae and an adjuvant. The present invention further relates to a method of using the antigen composition of the present invention for vaccinating an animal, preferably a mammal or a bird. In particular, the invention relates to pigs, lambs, dogs,
It relates to a method of vaccinating a horse, cow or human with the antigen of the present invention.

[0006] In one embodiment, E. coli. The stabilized antigens from the fluid fraction of Luciopathiae cultures are described. In one aspect, the stabilizer is E. coli. It is added to the supernatant or filtrate of the Luciopathiae culture, preferably to the concentrated supernatant or filtrate. Stabilizers can adsorb antigens. Non-limiting examples of stabilizers are aluminum hydroxide gel, aluminum phosphate gel, calcium phosphate gel, zinc hydroxide / calcium hydroxide gel and alum. In a preferred perspective,
The aluminum hydroxide gel is added to the concentrated supernatant to a final concentration of about 10% by volume of the aluminum hydroxide gel (ie,
(Volume of 10% by volume obtained by mixing 1 volume of the aluminum hydroxide gel with 1 volume of the supernatant) to about 40% by volume, more preferably about 30% by volume.

[0007] In another aspect, the concentrated E. coli. An antigen preparation comprising a Luciopathiae culture supernatant or filtrate and a stabilizing agent, when formulated for use as a vaccine preparation, is about 10- to 30-fold, preferably about 20-fold.
A 1: 2 dilution, thus reducing the concentration of the stabilizer in the vaccine formulation to less than about 5% by volume.

[0008] In another embodiment, E. Luciopathiae is cultured and processed to obtain E. coli. A supernatant or filtrate comprising the Luciopathiae antigen is obtained. In one aspect, E.I. Luciopathiae cultures are inactivated, for example by the addition of formalin or beta-propiolactone. In a further aspect, E.I. The Luciopathiae culture is separated from the bacteria, for example by centrifugation. In another further aspect, the supernatant is concentrated about 10-fold, for example, by molecular filtration.

In another embodiment of the invention, a preservative, such as merthiolate, is added to ethylenediaminetetraacetic acid (EDT).
Add to or with A) with or without A). In a further embodiment of the invention, the antigen of the invention is added to an adjuvant, such as an adjuvant comprising lecithin, oil and one or more surfactants. In a further aspect of the invention, a method of using the antigens and vaccines of the invention for vaccination of animals is described.

The present invention relates to compositions and methods for preventing or inhibiting erysipelas. In one aspect, the invention relates to E. coli. The present invention relates to an antigen of Luciopathiae and a method for producing such an antigen. The invention further relates to a vaccine formulation comprising the antigen of the invention. The present invention further provides E. coli for vaccination of animals, preferably mammals or birds. The present invention relates to a method of using an antigen of Luciopathiae.
In a most preferred aspect, the mammal is selected from the group consisting of pigs, lambs, dogs, horses, cows or humans.

The present invention relates to E. It relates to antigens obtained from Luciopathiae cultures. E. FIG. Any strain of Luciopathiae, such as the strain described in US Pat. No. 5,625,038, may be the source of the antigen of the invention. An isolable culture of the antigen can be provided in various ways. For example, the culture may be pure or substantially pure. More preferably, the antigen of the invention is E. coli. It is obtained from the supernatant or filtrate of the Luciopathiae culture. In a most preferred embodiment, the antigen of the invention is E. coli. Obtained from the supernatant or filtrate of a pure or substantially pure liquid culture of Luciopathiae.

E. Luciopathiae may be cultured by various methods known in the art. U.S. Patents 5,625,038; 5,616,328; 5,22 discussing bacterial culture
See 5,194; 4,981,685. For example, E. Luciopathiae may be cultured as described in the Examples below. E. FIG. Lucio Pasier is Sawada and Takahash
i, 1987, Am, J. Vet. Res. 48 : 239-242 and Groschup
1991, Epidemiol. Infect. 107: 637-649. General background on prokaryotic cell culture and processing is incorporated herein by reference. Maniatis, et al., 1982, Molecular.
Cloning, A Laboratory Manual , Cold Spring Harbor L
aboratory Press, Cold Spring Harbor, NY; Ausubel,
1989, Current Protocols in Molecular Biology , Gre
ene publishing Associates and Wiley Interscience, N
Y; Sambrook et al. 1989, Molecular Cloning, A Laborator
y Manual , 2d.ed., Cold Spring Harbor Laboratory P
ress, Cold Spring Harbor, NY.

In a preferred embodiment, the culture is inactivated by formalin (final concentration of about 0.5% by volume). In another preferred embodiment, the antigen of the invention is E. coli. Obtained from the supernatant or filtrate of the Luciopathiae culture. In a preferred embodiment, the culture supernatant or filtrate is concentrated about 10-fold and aluminum hydroxide (preferably REHYDRAG
EL ™) is added to the concentrated supernatant or filtrate to a final concentration of about 30% by volume to stabilize the antigen. In another preferred embodiment, a vaccine formulation comprising the antigen and an adjuvant is formulated, wherein the adjuvant comprises, for example, about 25% by volume of the vaccine formulation. In another preferred embodiment, thimerosal (about 0,1).
01% by volume) and EDTA (about 0.07% by volume)
Is added to this antigen as a preservative. A suitable adjuvant, referred to herein as "No. 1 adjuvant", comprises about 2% by volume of lecithin, about 18% by volume of mineral oil and about 8% by volume of a surfactant (eg, about 5.6% by volume of T
Ween 80 and about 2.4% by volume of Span 80) and the remaining volume is saline solution (eg Dulb)
ecco PBS). This adjuvant is incorporated herein by reference January 29, 1999.
It is described in a U.S. patent application filed daily.

E. Inactivation of Luciopathiae The antigen of the invention is E. coli . Obtained from Luciopathiae, which can be provided by means known in the art, for example, in liquid culture.
In a preferred embodiment of the present invention, the isolated E. coli from which the antigen is isolated. Luciopathiae cultures are inactivated before use as antigens in vaccine formulations. In the most preferred embodiment,
E. FIG. The Luciopathiae culture is inactivated before the liquid fraction is separated from the bacteria. E. FIG. Inactivation of the Luciopathiae culture is performed for various purposes, for example for sterilization or for inactivation of proteases or for preservation of antigen.

The culture containing the antigen of the present invention can be inactivated by various means known in the art. For example, the culture may be treated with an inactivating agent, ie, E. coli. Luciopathie may be exposed to a reagent that can sterilize it. Inactivating agents useful in the practice of the present invention induce an immune response in the animal and allow the animal to be protected from erysipelas. Deactivators known in the art, such as formalin (formaldehyde), beta-propiolactone or other chemicals having properties similar to these reagents may be used. Chemicals suitable for inactivating bacteria include, for example, contacting the bacteria with a particular chemical so that the bacteria are killed and whether the antigen is still effective for the ability to produce protective antibodies. The determination can be made by those skilled in the art, for example, by determining via mice vaccinated with the treated bacteria. See also U.S. Patent No. 5,225,194 for bacterial inactivation.

E. In a preferred embodiment, the antigens of the present invention are E. coli. Obtained from the fluid fraction of Luciopathiae culture. E. FIG. Luciopathiae may be cultured and the bacteria may be separated from the culture by, for example, centrifugation or filtration of the liquid culture. E. coli useful for the isolation of the antigens of the present invention. A culture of Luciopathiae can be provided by any method known in the art. For example, E. Luciopathiae may be grown in culture or medium to allow the bacteria to grow rapidly, ie, to the exponential growth phase. In a preferred embodiment, the culture utilized for the preparation of the antigen of the present invention has a logarithmic growth phase, when the treatment of the culture is started,
More preferably, it is in the late logarithmic growth phase.

In a preferred embodiment, E. The Luciopathiae culture is processed to separate all or substantially all of the bacteria from the culture or medium in which they were grown. For example, about 9
0%, more preferably about 95%, more preferably about 98% of the bacteria may be removed from the culture or medium. E. FIG. Luciopathie can be separated from the culture broth or medium by any means known in the art. For example,
E. FIG. Luciopathiae may be centrifuged to separate the bacteria from the culture or medium. E. FIG. Any centrifugation known in the art that is capable of sedimenting Luciopathiae is suitable for separating cells from the culture or medium. For example, continuous flow centrifugation can be utilized. Background on how to remove bacteria from the culture medium can be found in Maniatis, et al. 1982, Molecular Cloning, A Laboratory.
Manual , Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY; Ausubel, et al. 1989, Current Proto
colsin Molecular Biology , Greene publishing Associ
ates and Wiley Interscience, NY; Sambrook et al. 1989,
Molecular Cloning, A Laboratory Manual , 2d.ed., Co
ld Spring Harbor Laboratory Press, Cold Spring Har
bor, NY.

In another aspect, E.I. Lucio Pasier,
The bacteria or bacteria may be retained but the antigen of the invention may be removed by filtering the culture or medium through a filter that cannot. Numerous filters suitable for separating bacteria from antigens in cultures or media are known in the art. For example, filters useful for separating bacteria from the fluid fraction have an average pore diameter of about 0.1 micron to about 0.5 micron, more preferably about 0.2 micron.

The above E. The fluid fraction obtained from the culture of Luciopathiae may be concentrated. In one embodiment, the fluid fraction is about 3-30 times, for example, about 3 times, or about 6 times,
Or about 10 times, or about 15 times, or about 20 times, or about 3 times
It may be concentrated 0 times. This fluid fraction may be concentrated by any means known in the art. In a preferred embodiment,
This fluid fraction may be concentrated using hollow fiber filtration. In one aspect, hollow fiber filtration is about 5,
000 kilodaltons to about 50,000 kilodaltons, more preferably about 10,000 kilodaltons to about 30,0 kilodaltons
It is carried out with a molecular weight cut-off of 00 kilodalton. U.S. Pat. No. 5,22, discussing concentration of the fluid fraction of a bacterial culture
See also 5,194.

The fluid fraction may be concentrated by freeze-drying. In another aspect, the fluid fraction may be concentrated by precipitation of the proteins and polypeptides in the fluid fraction, followed by resuspension of the precipitate. The protein can be precipitated from the fluid fraction via any method known in the art, for example, precipitation with polyethylene glycol, ethanol or ammonium sulfate. After precipitation, the precipitate may be resuspended in any solution suitable for the preparation of a vaccine formulation, for example, a saline solution.

E. Stabilization of Luciopathiae antigen Antigens obtained from the fluid fraction of Luciopathiae cultures are effective immunogens for preventing or suppressing erysipelas in animals. However, the stability of such antigens after bacterial removal is a serious problem when using such antigens in vaccine formulations. The present invention solves this problem and, in part,
E. FIG. It is based on the discovery that the antigen in the fluid fraction of a Luciopathiae culture can be stabilized by the addition of a stabilizer.

Any stabilizer known in the art can be used to stabilize the antigen of the present invention. In a preferred embodiment, the stabilizer is E. coli. It is possible to adsorb antigens of the Luciopathia culture fluid fraction. Suitable stabilizers are described in E.I. The antigenic capacity of the fluid fraction of the Luciopathiae culture can be maintained, or the loss of its antigenic capacity after bacterial removal can be delayed. The stabilizing effect of such a reagent can be determined by experiment. For example, inactivated E. coli. Two samples of the fluid fraction of the Luciopathiae culture, one containing the chemical to test its use as a stabilizer, and the other without it at 37 ° C. for a predetermined period of time, for example from about 14 to about 28 days Incubation may be performed. These samples were then subjected to a standard mouse efficacy test (9CFR 113.119).
According to (c)), the test is carried out with a vaccination of mice using an adjuvant, for example a No. 1 adjuvant. A higher percentage of protected animals in a given group of vaccines treated with such chemicals than in a given group of untreated vaccines or control animals without vaccination was due to the antigen in the chemically treated vaccines. Suggest that was stabilized.

The above experiment illustrates an accelerated stability test at a higher temperature (37 ° C.) than is normally utilized for storage. Usually, the antigen preparation is stored at a low temperature, for example, from about 2C to about 8C. Stability at 37 ° C. for 28 days suggests stability over a longer period of normal cryopreservation, ie, over a period of several years. In one embodiment, the antigen is stable according to the present invention at low temperatures for up to about 5 years, more particularly at low temperatures for up to about 3 years. In another embodiment, the antigen is stable at low temperatures for at least one year according to the invention.

Various antigens known in the art that can adsorb antigens, such as aluminum hydroxide gel,
Aluminum phosphate gels, calcium phosphate gels, zinc hydroxide / calcium hydroxide gels and alums (eg potassium alum) are useful as stabilizers. In a preferred embodiment, an aluminum hydroxide gel, such as REH
YDRAGEL ™ is used as a stabilizer (US Pat.
616,328 and 5,232,690).

In one embodiment, a metal hydroxide gel, such as an aluminum hydroxide gel (eg, REHYDRAG)
EL) in the antigen formulation from about 10 to about 40% by volume, such as about 10% by volume, or about 20% by volume, or about 30% by volume;
Alternatively, add to a final concentration of about 40% by volume. In a preferred embodiment, the aluminum hydroxide gel (eg, REHYDRAGEL) is added to the antigen formulation at about 30% by volume.
To a final concentration of.

The antigen preparation containing the stabilizer is used to formulate a vaccine preparation, for example, by adding an adjuvant and a diluent, for example, saline, to dilute the antigen preparation and the stabilizer. Good. Such dilution may be useful to avoid or substantially avoid unwanted side effects of the vaccine formulation in animals. For example, adding a metal hydroxide gel, such as an aluminum hydroxide gel (eg, REHYDRAGEL), to a vaccine formulation can be about 5 times, or about 10 times, or about 15 times, or about 2 times.
Dilute 0-fold, or about 25-fold, or about 30-fold. In a preferred embodiment, an aluminum hydroxide gel (eg, REH
The antigen formulation containing (YDRAGEL) at a final concentration of about 30% by volume is diluted about 20-fold by adding to this vaccine formulation to a final aluminum hydroxide gel concentration of about 1.5% by volume.

E. Wak comprising an antigen of Luciopathiae
Chin formulation The antigens of the present invention can be used in vaccine formulations to immunize animals. In one embodiment, the vaccine formulation comprises an antigen of the invention and an adjuvant. In a preferred embodiment, an adjuvant useful for the vaccine formulation of the present invention comprises lecithin, an oil and a surfactant. Preferred adjuvanted formulated vaccine formulations are about 0.25 to 1
2.5% by volume, more preferably about 0.5 to about 5% by volume,
And most preferably about 0.5-1.25% by volume of lecithin, about 1-23% by volume, more preferably about 3.5-1% by volume.
0% and most preferably about 4.5% oil by volume;
And about 1.5 to about 6% by volume, more preferably about 1.5 to about 6% by volume.
It contains 4% by volume, and most preferably about 2% by volume of amphiphilic surfactant. Preferably, the adjuvant is 2
Types of amphiphilic surfactants, such as Tween and Sp
an surfactant, one of which is primarily present in the aqueous phase of the vaccine formulation (eg, Tween 8).
0), and the other is in the oil phase (eg, Span
80). Preferably, Tween 80 and Span
When 80 is used as a surfactant, Tween 80
Is about 1.5 to about 3 times, preferably about 2 times the concentration of Span 80. Suitable adjuvants are aqueous carrier solutions, such as phosphate buffered saline (PBS) (eg, Du
lbecco PBS). Lecithins and oils suitable for adjuvants for vaccine formulations are DRAKEOL
® is a mixture of lecithin in 5Lt mineral oil. Lecithin is available from Central Soya, Fort Wayne, Indiana. Tween and Span surfactants are Van Wate
Available from rs and Rogers, Omaha, Nebraska.

In another embodiment, adjuvants known in the art, such as US Pat. Nos. 5,846,527; 5,417,97, which discuss adjuvants.
1; 5,232,690, aluminum hydroxide, muramyl dipeptide, zinc calcium hydroxide, abridine, aluminum hydroxide, oil and saponin may be used in the vaccine formulation of the present invention.

A preferred vaccine formulation comprises about 10 to 50% by volume of the adjuvant composition, more preferably about 1% by volume.
5 to 35% by volume, more preferably about 20 to 30% by volume,
And it is most preferably prescribed in about 25% by volume.

The vaccine preparation may be administered to the subject as it is or in the form of a pharmaceutical or therapeutic preparation. Pharmaceutical preparations comprising the antigens can be prepared by conventional mixing, dissolving, comminuting, sugar-coating, grinding, emulsifying, entrapping, entrapping or lyophilizing processes. Pharmaceutical formulations may be prepared by conventional means utilizing one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the antigen of the invention into a pharmaceutically usable formulation. It can be compounded by means.

[0031] Systemic formulations include those designed for administration by injection, eg, subcutaneous, intradermal, intramuscular, or intraperitoneal injection.

For injection, the antigens may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, phosphate buffered saline, or any other saline. . The solution may include formulating agents such as suspending, stabilizing and / or dispersing agents.
Alternatively, the protein may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

In addition to the above formulations, the antigens may be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the antigen may be incorporated into a suitable polymer or hydrophilic material (eg, as an emulsion in an acceptable oil) or ion exchange resin,
Alternatively, they may be formulated as a slightly soluble derivative, for example, a slightly soluble salt.

On the other hand, other drug delivery systems can be employed. Liposomes and emulsions are well known examples of delivery vehicles that can be used to deliver antigen. Certain organic solvents, such as dimethylsulfoxide, may be utilized, but usually involve significant toxicity. Alternatively, the antigen may be introduced utilizing a sustained release system, for example, a semi-permeable matrix of a solid polymer containing a therapeutic or vaccinating agent. Various release materials have been established and are well known to those skilled in the art. Retained-release capsules may release the antigen for several weeks to over 100 days, depending on its chemical nature. Depending on the chemical nature and biological stability of the reagent, further strategies for antigen stabilization may be employed.

Determining an effective amount of an antigen for administration is within the ordinary ability of those skilled in the art, especially with reference to the disclosure herein.

The effective dose can be estimated initially from in vitro assays. For example, dosage forms can be formulated in animal models using techniques well known in the art to achieve the induction of an immune response. One skilled in the art can readily optimize administration for all animal species based on the results described herein. For example, when used as a vaccine, the antigen of the present invention may be
About 1 to about 3 doses may be administered over a 36 week period. Booster doses may be given periodically thereafter. Other protocols may be appropriate for individual animals. A suitable dose is that when administered as described above, the animal is administered to E. coli. An amount of antigen capable of raising an immune response in an immunized animal is sufficient to provide an immune response sufficient to protect against Luciopathiae infection for at least 4-12 months.

The amount of antigen in the dosage form is the optical density (E ₆₂₅ ) of the culture at inactivation and is specified as opacity units.
If the optical density at inactivation is 4.0, for example, 1 ml of culture supernatant or filtrate prepared from this culture contains 4 opacity units, 0.5 ml contains 2 opacity units, and so on. It has nothing to do with removing the bacteria that are the source of opacity. If, for example, a supernatant fluid containing 5 opacity units per ml is concentrated 12-fold by molecular filtration, the concentrated fluid will have a value of 60 opacity units per ml. Generally, the amount of antigen in a vaccine dosage form will be from about 1 to about 12
Opacity units, preferably in the range of about 2 to about 4 opacity units. Suitable dosage forms will vary depending on the route of injection and the size of the host, and will typically be from 0.1 to about 5 ml. Preferably, when the host is a pig, the pig is at least two weeks old.

[0038]

EXAMPLES The vaccinia study presented below shows that the vaccine described herein having the No. 1 adjuvant was administered to piglets about 3-6 weeks of age in a two dose regimen to piglets during the second dose. It has been demonstrated that significant protection against virulent load is provided 20 weeks after vaccination. The antigen with the No. 1 adjuvant also induces a substantial antibody response 2 weeks after vaccination, as shown by serum ELISA, but upon challenge these titres are reduced and thus the titer of the individual Was not closely correlated with protection. Similarly administered antigen in saponin adjuvant induced lower ELISA titers and proved unsuitable for protecting pigs from virulent challenge 20 weeks after the second vaccination.

The loading model was very effective in that 10 control pigs showed clinical signs of erysipelas after loading (Table 2). Three pigs met the high temperature criteria and the other five were E. coli. Luciopathie strain was positive. The remaining two control pigs did not meet any of the above criteria. However, they have been sick for several days and have metastatic skin damage that is characteristic of the disease and thus killed humanely. In contrast, N
o. In the group vaccinated with the vaccine containing one adjuvant, 15 out of 20 pigs were completely protected (Table 3).

The E. coli of the present invention with No. 1 adjuvant An experimental vaccine containing the Luciopathiae antigen, when given to piglets about 3-6 weeks old in a two dose regimen, has a significant effect on the development of clinical erysipelas due to virulent loading 20 weeks after the second vaccination. Provided protection. See Table 3 below. In addition, a similar experimental vaccine with saponin adjuvant, when given to piglets according to the same regimen, has
No protection was provided against the development of clinical erysipelas by virulent loading 20 weeks after the second vaccination. Finally, vaccinia containing the antigen of the present invention, regardless of the adjuvant used, induced a substantial serum response that peaked two weeks after the second vaccinia.

The present invention is further described by the following non-limiting examples.

Embodiment 1 E. FIG. Luciopathiae culture Luciopathiae strain CN 3342 was purified from Difco Proteose Peptone, Difco Y at a concentration of 2.75% in deionized water.
east Extract (0.55%), Tween 80 (0.2%), K ₂ HPO ₄
(0.217%) and KH ₂ PO ₄ (0.061%). The pH of this medium is adjusted to 7.2 with 5N NaOH. The medium is steam sterilized at a minimum of 122 ° C. for 30-90 minutes. After autoclaving, a sterile 50% dextrose solution is added to a final concentration of 3% w / w.

A working seed culture is obtained by removing a frozen test tube of a master seed specimen from a frozen stock (−70 ° C.), rapidly thawing, and aseptically transferring the contents to a medium flask. Prepare. The flask is incubated for 12-36 hours at 37 ° C. with shaking and assayed for purity by Gram stain. If found to be pure, the culture is mixed with sterile glycerin (10%), dispensed in 1 ml aliquots into cryotubes and stored frozen.

The production medium (production med)
ium) is inoculated with 0.01 to 2% of the master or effective species. When using a seed fermenter containing 10-100 liters of production medium, 1-5% from the seed flask
Inoculation with the culture of In a production fermentor containing 200 to 10,000 liters of production medium, 0.5
Inoculate ~ 5% culture.

The production culture is incubated with agitation at a set point of 37 ± 2 ° C. Incubation times may vary from 4 to 24 hours. The pH is maintained at 7.2 ± 0.1 by adding 10N sterile sodium hydroxide solution to the culture during the incubation time. During the growth phase, a regular addition of dextrose is made.

Prior to harvest, the culture is examined microscopically for purity, morphology and Gram reaction. The growth is 6
Follow by measuring the optical density at 25 nm. 6 cultures
Collected when having an optical density of 4.0 or more at 25 nm.

Embodiment 2 FIG . E. FIG. Preparation of Luciopathie vaccine
A formalin solution was added to the culture to a final concentration of 0.5% (v / v) to inactivate the culture.
Transfer the culture to a sterile tank and place in a 37 ± 2 ° C. incubator for a minimum of 24 hours (and a maximum of 60 hours)
Placed under constant stirring. Cultures were not processed immediately, but were stored at 2-8 ° C for up to 7 days. Inactivated cultures were clarified via continuous flow centrifugation. The fluid fraction was retained for further processing and the bacteria were discarded.

In the experiments, a 10 × concentration was applied to a final concentration of 0.1% by volume of beta propiolactone (“BP
L)) or inactivated with either 0.2% by volume final concentration of formalin (> 24 h at 37 ° C.) Performed on filtrates of Luciopathiae cultures. E. FIG. A 64-66 kD protein found in the culture filtrate of Luciopathiae (Gros
chup et al., 1991, Epidemiol. Infect. 107: 637-649 and U.S. Pat. No. 5,625,038).
Inactivation and stabilization effects were determined based on the presence of the 6 kDa protein. Consistent with the previous finding that formalin inactivation of the culture reduced the ELISA assay of the protein, the BPL concentrate was about 4% less than the formalin concentrate.
It had twice the assay value. After incubation at 37 ° C. for 14 days, the BPL concentrate lost about 80% of its value compared to about 40% for the formalin concentrate. However, in both cases, 3
Pre-addition of 0% by volume of REHYDRAGEL almost prevented the loss and retained virtually all of the formalin formulation, as shown below. A small test in pigs showed that the fluid fraction of the formalin-inactivated culture was higher than that of the BPL-inactivated culture, and the virulent E. It has been shown to be effective in protecting pigs against load by Luciopathiae.

The fluid was concentrated to 6 × to 20 × (usually about 10 ×) by hollow fiber filtration after centrifugation (apparent molecular weight cutoff of 10,000 kDa). The fluid was stabilized after concentration by adding aluminum hydroxide gel.

To stabilize the immunogen, the aluminum hydroxide gel was added to the concentrated fluid while stirring to a final volume of 30% by volume.
(30 volumes of gel versus 70 volumes of concentrate). 20 of this concentrate in the vaccine
After doubling dilution, the Al gel content was only about 1.5% and was not sufficient to produce a negative reaction at the injection site. 1
0-fold concentrated E. coli. Titration to determine the amount of Al gel needed to adsorb all the protected proteins in the Luciopathiae culture fluid fraction was greater than 95% by 32% by volume of REHYDR.
AGEL (Reheis, Berkley Heights, New Jersey). Thimerosal (ie MERT
HIOLATE (trademark)) (Dimportex, Spain; Flavine
Inc., Klosters, New Jersey) was added as a preservative to this product to a final concentration of about 0.01% by volume. The concentration of thimerosal in the vaccine composition containing the antigen composition and the antigen of the present invention is about 0.01 w /
v. EDTA (Sigma, St. Louis, Missouri)
Was added to a final concentration of about 0.07% (w / v).

The adjuvant used was No. 1 adjuvant. 200 ml of 1000 ml of No. 1 adjuvant
Filter sterilized lecithin-oil solution (DRAKEOL)
(Trademark) 10% lecithin in mineral oil), autoclaved Tween 80 (56 ml) and Span 80
(24 ml), and phosphate buffered saline (Dulbecc)
oPBS) (720 ml). The lecithin-oil solution and Span 80 were combined and mixed in a sterile tank at room temperature for at least 1 hour until emulsification was complete. The saline and Tween 80 were combined and mixed for at least 1 hour at room temperature in a sterile tank. The oil mixture was emulsified with the aqueous mixture using a Ross emulsifier. Emulsification continued by recirculation until all adjuvant was added into the saline solution. This emulsion is Gou
The lin press was applied twice at room temperature. This adjuvant was stored at 2-8 ° C.

A 5 L vaccine was made by adding 1 L of adjuvant to 3 L of aqueous phase. This aqueous phase comprises sufficient stabilized concentrate to give a final antigen content of 3.2 opacity units per 2 ml dose of vaccine, and enough saline to make the volume 5 L. . The opacity unit is defined as (optical density at recovery (E ₆₂₅ ) × concentration factor).

Embodiment 3 FIG . Pig E. Luciopathie vaccinia
And load E. The eighth subculture (MS + 8) of Luciopathiae strain CN3342 was used for vaccine production. One dose level of stabilization, purification, calculated from the optical density (OD) of the culture at inactivation. Luciopathie antigen (3.2 opacity units [OU]) was utilized. One OU corresponds to one ml of fluid having one OD (determined at 625 nm). No. 1 adjuvant or saponin (0.05 w / v; Berghansen Chemica
l Company, Cincinnati, Ohio) was used as adjuvant. For details, see E. Luciopathie strain CN
The 3342 culture was grown to an OD of 5.28. This culture was inactivated with 0.5% formalin for 24 hours. After inactivation, the bacteria were removed by centrifugation. The fluid fraction was concentrated using an ultrafiltration unit with an apparent molecular weight cutoff of 10,000 kDa. The fluid fraction was concentrated about 13.4 times. The antigen is added to REHYDRAGEL (30
% By volume). The adsorbed concentrate was stored at 4 ° C. until vaccine formulation. The vaccine was formulated with No. 1 adjuvant or 0.05% saponin as adjuvant. The stabilized antigen was diluted in saline (150 mM sodium chloride and 4 mM phosphate) to a final concentration. Ethylenediaminetetraacetic acid (EDTA, 0.0
7%) and Tylomesal (0.01%) were added to the final vaccine formulation. Phosphate buffered saline was used as a placebo. Table 1 summarizes treatment groups, vaccine treatments, and the number of piglets that received vaccination and challenge.

[0054]

[Table 1] Satisfactory high body temperature (4 for at least 2 consecutive days)
0.9 ° C. (105.6 ° F. or higher), culture at necropsy, and / or E. coli. Characteristic clinical signs of infection by Luciopathiae were manifested in control animals. Clinical signs that may be characteristic of the disease include sudden death, weakness, abdominal and ear congestion, metastatic skin damage,
And joint or joint involv
element). Pigs with clinical signs but below the body temperature standard are sacrificed and the blood, spleen and liver are cultured to give E. coli. An attempt was made to isolate Luciopathie.

Fisher's extraction test was used to determine if there was a difference in the percentage of animals protected with the various vaccines (p <0.05). To compare the different treatment groups with the control, and to compare each group with the mean of all other treatment groups,
A prior contrast was constructed. Using logistic regression, the type of vaccine given and its relationship to the serological response of each group of piglets were analyzed at 2 months, 3 months, 4 months, 5 months, and prechallenge pigs.
Performed on blood. The relationship between vaccine-induced titer at loading time and disease status (protected / unprotected) was assessed using logistic regression. A significance level of 5% was used for a true relationship declaration.

E. Low against Luciopathie (# 80
0) 20 pregnant sow / ELISA serum titers /
Young sows were obtained from Riddell Farms, Albert City, IA, and the University of Nebraska Department of Vet
They were kept in erinary and Biological Sciences isolation rooms.

ELISA Serum titers were determined by whole cell direct antigen binding ELISA as follows. Preparation of antigen-coated plate and ELISA using such plate
See U.S. Pat. No. 4,918,163 to A. First, E.I. Luciopathiae was grown as described in Example 1 and recovered from exponential phase cultures.
The optical density at 640 nm was recorded and converted to cell number / ml using a table established via bacterial counts from solutions with different optical densities. Live bacteria in PBS
(Dulbecco PBS, Sigma, St. Louis, Missouri) diluted to a density of about 1.1 × 10 ⁹ cells / ml. This P
Live bacteria diluted in BS were bound to ELISA plates. For plate preparation, 100
μl of 0.1% by volume glutaraldehyde (Sigma, St.
(Louis, Missouri) was added to each well, the wells were covered, and the plates were incubated at 37 ° C for 1 hour. Glutaraldehyde in PBS was removed from each well, and the wells were dried with an absorbent towel. About 1.1 × 10 ⁹ cells / in PBS
100 μl of live bacteria at a density of ml was added to each well.
The plates were centrifuged at 2000pm for 5 minutes at 22 ° C. Then 200 μl of PBS (PVA / PBS)
1% of polyvinyl alcohol in Aldrich, Milwauke
e, Wisconsin) was added to each well, the wells were covered, and the plates were left overnight at 4 ° C. The contents of the wells of the plate were transferred to a sterile solution and the wells were washed with PBS. The wells were covered with gauze and allowed to dry at room temperature (about 1 hour).

The ELISA procedure was performed using plates with bound bacterial whole cell pathogen as follows.
First, porcine serum containing the positive control was 1% PV
A / PBS was diluted. All unknown sera were diluted 1:50 and a positive control was used at 1: 200. 200 μl of each sample was added to the wells in row A. 100 μl of 1% PVA / PBS was added to the remaining wells. Column B with 2-fold serial dilutions based on each sample
To H. Cover the wells and 37
Incubated for 1 hour at ° C. The wells were then washed three times with PBST. 100 μl of a 1: 2000 dilution of goat anti-swine IgG (H and L) peroxidase conjugate prepared in 1% PVA / PBS (Ki
rkegaard and Perry, Gaitherburg, Maryland) was added to each well. The wells were re-covered and incubated at 37 ° C for 1 hour. These wells were washed three times with PBST. 100 μl of ABTS substrate (2,2′-azino-di- (3-ethylbenzthiazoline sulfonic acid) obtained from Kirkegaard and Perry, Gaitherburg, Maryland) was added to each well and the plates were incubated at room temperature for 10 minutes. did. The plates were shaken on a microplate shaker for 10 seconds, then the absorbance of each well was measured at 405-490 nm using a plate reader black. The endpoint titer of each unknown serum is that its absorption is 1: 320
The serum dilution was greater than the absorption of the positive control at a dilution of 0.

The sow is bled 0-10 days before parturition,
The E. Luciopathie antibody titers were determined. Piglets were randomly divided based on the serum titer of the sow and the date of delivery.
Blood was collected from 58 piglets from these sows / young sows and two experimental E. coli. Vaccinations were given about 3 weeks after birth with either one of the Luciopathie vaccines or a placebo (groups shown in Table 1). At about 4 weeks of age, the piglets weaned. At approximately 6 weeks of age, piglets were bled and revaccinated with the same vaccine. Blood was collected from all pigs at about 2, 3, 4 and 5 months of age. At about 5.5 months of age, all lean pigs were removed from the study. At about 6 months of age (20 weeks after the second vaccination), pigs were bled and 40 pigs were grown from cultures administered by the National Veterinary Services Laboratory. 2 ml of virulent culture of Luciopathiae (237 mouse LD ₅₀ , 1.74
(× 10 ⁹ colony forming units / ml). Animals were followed for signs of clinical disease and rectal temperature 2 days before challenge, the day of challenge, and 7 days after challenge. Any control animals meeting the criteria for hot rectal temperature (40.9 ° C) were removed from the study and treated with injected penicillin. Any control animals that have clinical signs of illness but do not meet the hot rectal temperature criteria are humanely killed, necropsied, and whole blood, spleen and liver samples are collected from E. coli. Cultured for Luciopathiae. Any dead control animals are necropsied and spleens and livers are isolated from E. coli. Cultured for Luciopathiae. Any vaccinated animals meeting the criteria for elevated rectal temperature (40.9 ° C.) and / or clinical signs of illness were removed from the study and treated with injected penicillin. Any vaccinated animal that died after challenge was necropsied and spleen and liver samples were collected from E. coli. Cultured for Luciopathiae. E. FIG. Antibody titers to Luciopathiae were determined by ELISA as described above and a correlation between antibody titers and clinical protection was performed.

[0060] ELISA titers were determined for serum from a single blood sample from a sow and all blood samples from seven pig collection periods. Aerobic bacterial cultures (48 hours, 37 ° C., blood agar) were performed on blood and / or spleen and liver samples from pigs that had died or were killed by lethal injection.

Results E. coli control pigs Table 2 shows the results of Luciopathie load
Put together. All ten pigs were positive for erysipelas.

[0062]

[Table 2]

Vaccine with No. 1 adjuvant (TO
E. of the pig given 2). The results for pigs loaded with Luciopathie are summarized in Table 3. 15 out of 20 pigs (75
%) Was completely protected.

[0064]

[Table 3]

Vaccines with saponin adjuvant (T
03). The results of Luciopathie loading are summarized in Table 4. Only one pig was protected.

[Table 4]

Geometric mean ELISA for piglet groups
The titers ("GMT") are shown in Table 5.

[0067]

[Table 5]

Piglets have very low E. coli at the time of the first vaccination. It had an antibody titer specific for Luciopathiae. These titers ranged from 50 to less than 200. In control piglets, GMT increased slightly during the study and ELI
It suggested that SA was not very specific to aged pigs.

Vaccines with either No. 1 adjuvant or saponin as adjuvant receive a statistically significant (p = 0.0001) serum peak at 2 weeks after the second vaccination (about 2 months after birth). A response was induced. The titers of both groups continued to decline over time (up to about 5 months of age), and GMT of piglets that received the antigen in No. 1 adjuvant at all time points was lower than that of piglets that received the antigen in saponin adjuvant. It was clearly higher than GMT. At loading, the GMT of pigs receiving the antigen in No. 1 adjuvant was slightly higher than twice that of the control, while the GMT of pigs receiving the antigen in saponin adjuvant was slightly lower than twice the control. Protection from clinical disease after challenge did not correlate with individual ELISA titers (p> 0.05). However, an interesting observation was the peak titer observed two weeks after the second vaccination in pigs that had received the antigen in No. 1 adjuvant. No. 1 Vaccinated with antigen in adjuvant 2
Of the 0 piglets, 8 had a peak titer of 2800 or more (8/8 protected from load) and 8 had a peak titer of 6400 (6/8 protected from load). And 4 had 3200 peaks (1/4 protected from load),
A titer of 6400 or more suggested that it was an indicator of long-term protection.

In summary, ten vaccinated controls were infected. In the group of pigs that received the vaccine with the No. 1 adjuvant, 15 out of 20 were protected. In the group of pigs that received the vaccine with saponin as adjuvant, only one in ten was protected.

Embodiment 4 FIG . Antigen Stabilization by Al Gel Vaccines were prepared as described in Examples 1-3, including treatment of the antigen with Al gel as described above. The vaccine was tested for efficacy in pigs. Pigs were vaccinated by two 2 ml doses given intramuscularly (IM), the first dose being about 3 weeks (weaning), and the second dose 3 weeks later. Controls received phosphate buffered saline as placebo. At about 9 weeks of age, virulent E. A load of 1 M injection of Luciopathiae was applied.
As shown in Table 6, protection from vaccination was 100% at 9 weeks.
It became. This vaccine is already available for vaccination of pigs.
It was two months old. This result confirms that the protective antigen was effectively stabilized.

[0072]

[Table 6]

Remarks: No. 20 pig was excluded. It was a very wild animal, and was so violent that it couldn't measure its temperature. After the load, the pig remained completely healthy.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Leroy Allen Swarelingin Connecticut, United States 06385, Waterford, Volkshall Street Extension 653 (72) Inventor Brian Thomas Sweeter United States, Nebraska 68516, Lincoln, Plum Creek Drive 7425

Claims (11)

[Claims] 1. An antigen composition comprising a fluid fraction derived from a culture of Erysipelothrix rhusiopathie (Erysipelothrix rhusiopathie) and a stabilizer. 2. The method according to claim 1, wherein the stabilizer is a metal hydroxide, metal phosphate, aluminum hydroxide gel, aluminum phosphate gel, calcium phosphate gel, zinc hydroxide / calcium hydroxide gel or alum. An antigen composition. 3. The antigen composition according to claim 1, wherein the culture of E. persieryx luciopathiae is inactivated. 4. The antigen composition according to claim 3, wherein the culture of E. persicum lusiopaciae is inactivated with formalin or beta-propiolactan. 5. The antigen composition of claim 1, wherein said fluid fraction is about 3-30 fold concentrated. 6. A vaccine preparation comprising the antigen composition according to claim 1 and an adjuvant composition. 7. The method of claim 6, wherein the adjuvant composition comprises about 0.25
7. The vaccine formulation of claim 6, comprising 12.5% by volume of lecithin, about 1 to 23% by volume of oil, and about 1.5 to 6% by volume of amphiphilic surfactant in the vaccine formulation. . 8. A method for producing an antigen composition, comprising adding a stabilizer to a fluid fraction derived from a culture of E. persicum L. luciopathiae. 9. A method of vaccinating an animal, comprising administering an effective amount of the antigen composition of claim 1 to an animal other than a human. 10. The method of claim 9, wherein the animal is a pig. 11. A method of vaccinating an animal, comprising administering to the animal an effective amount of the vaccine preparation of claim 6.